The target reaction in the antiviral action of mouse interferon against vesicular stomatitis virus multiplication

Treatment of mouse L929 cells with mouse interferon (IFN) lowered the yield of vesicular stomatitis virus (VSV) in a dose-dependent manner. Accumulation of viral proteins was severely inhibited in IFN-treated cells, whereas cellular protein synthesis was not, indicating that the virus-induced shutoff of cellular protein synthesis was prevented by IFN. In order to identify the major target of IFN action precisely, the effect of IFN treatment on the synthesis of viral RNAs and proteins at various stages during the course of viral replication was examined. Accumulation of viral RNAs late in infection was inhibited, as was the case with viral proteins, but the synthesis of leader RNA and mRNAs early in infection was not significantly inhibited by treatment with a moderate dose of IFN. On the other hand, viral protein synthesis at an early stage of infection was strongly inhibited by IFN. The results indicate that the major target reaction of antiviral action of IFN against VSV multiplication is the translation of viral mRNA.     

Mass spectrometry reveals specific and global molecular transformations during viral infection

Mass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.     

与PRRSV nsp11互作的宿主细胞蛋白鉴定及生物信息学分析

【目的】研究猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)nsp11与宿主细胞蛋白之间的相互作用,对于揭示nsp11在病毒复制过程中发挥的功能至关重要。【方法】在病毒感染细胞的基础上,利用nsp11的单克隆抗体,采用免疫沉淀结合串联质谱的方法,筛选与PRRSV nsp11相互作用的宿主细胞蛋白,并对所筛选出的宿主细胞蛋白进行了GO注释、COG注释和KEGG代谢通路注释;选取筛选出的宿主细胞蛋白IRAK1,利用免疫共沉淀技术和激光共聚焦技术鉴定其与nsp11之间的相互作用。【结果】与空白对照组相比,病毒感染组中出现3条差异带;经质谱分析共筛选得到了201个与nsp11相互作用的宿主细胞蛋白,分别与蛋白质代谢、细胞信号通路转导以及病原致病性等密切相关;在生物信息学分析的基础上,实验验证了nsp11确与宿主细胞蛋白IRAK1进行相互作用。【结论】鉴定出与PRRSV nsp11相互作用的宿主细胞蛋白,生物信息学分析显示它们在病毒的复制和致病过程中发挥重要作用。研究结果为探究nsp11的生物学功能指明了方向,也为研究宿主细胞蛋白与病毒蛋白间的相互作用及其调控病毒复制和致病性的分子机制奠定了基础。     

Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome

Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.     

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

ABSTRACT: BACKGROUND: Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. RESULTS: Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3[PRIME] poly(A) sequence identifying the 3[PRIME] end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. CONCLUSIONS: NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein synthesis during infection.     

Interaction of virally coded protein and a cell cycle-regulated cellular protein with the bovine parvovirus left terminus ori.

Replication of parvoviruses requires cis signals located in terminal palindromes that function as origins of replication in conjunction with trans-acting viral and cellular proteins. A gel retardation assay was used to identify proteins in crude nuclear extracts of bovine parvovirus (BPV)-infected bovine fetal lung cells that interact with the hairpinned left end (3' OH terminus of the viral minus strand in the flop conformation) of BPV. Three specific DNA-protein complexes formed. One complex was shown to involve a BPV structural protein(s) by inhibiting its formation when antiserum specific for these BPV proteins was used. By specific competition with serum containing antibodies against the BPV nonstructural proteins, a second complex was shown to involve a BPV nonstructural protein. A third complex contained protein of cellular origin and was also formed with extracts of uninfected bovine fetal lung cells. DNA competition assays suggest that the viral proteins do not bind to the right hairpin, which differs in sequence and secondary structure from the left terminus, or to a BPV terminus that lacks the first 52 nucleotides, preventing formation of the stem of the hairpin. The cellular protein is regulated in a cell cycle-dependent fashion, with its binding activity increased in uninfected, actively dividing cells compared with contact-inhibited cells. Since autonomous parvovirus replication requires an S-phase factor for progeny formation, the terminal binding protein demonstrated here is a candidate for this factor.     

与猪流行性腹泻病毒M蛋白互作的宿主蛋白的鉴定

【背景】猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)膜蛋白(M)在病毒粒子的组装、膜融合和病毒复制等方面具有重要的作用,但M蛋白与宿主细胞的互作机制尚不清楚。【目的】利用免疫沉淀技术和液质联用技术筛选细胞内与PEDVM蛋白相互作用的蛋白,为揭示M蛋白在病毒增殖过程中发挥的功能提供研究基础。【方法】将MOI=0.1的PEDV DR13疫苗株接种于长成单层的Vero细胞,感染36 h后,收集细胞并进行裂解。利用抗M的单克隆抗体沉淀与M相互作用蛋白复合物,通过液相色谱串联质谱(LC-MS/MS)进行鉴定并利用细胞功能富集分析(Gene ontology,GO)对感染组鉴定到的细胞蛋白进行分析,确定两个细胞内源性蛋白为候选蛋白,进行免疫共沉淀(Co-IP)验证和共定位分析。【结果】基于鉴定蛋白的肽段数的方法分析显示,感染组与对照组相比,鉴定了218个与M蛋白相互作用的细胞内源性蛋白,分别与蛋白质合成、代谢、细胞信号通路转导等密切相关,选择细胞分裂周期蛋白42 (Cell division cycle 42,CDC42)、真核翻译起始因子3亚基L蛋白(eIF3L)为候选蛋白进行Co-IP(Co-immunoprecipitation)验证和共定位分析,结果证实CDC42、eIF3L蛋白分别与M蛋白在细胞内存在相互作用。【结论】鉴定出PEDV M蛋白能够与宿主细胞CDC42和eIF3L蛋白相互作用,并鉴定出其他可能与M蛋白发生相互作用的宿主蛋白60个,为开展PEDV与宿主细胞蛋白相互作用研究提供了重要理论依据。     

Proteomics of herpes simplex virus replication compartments: association of cellular DNA replication, repair, recombination, and chromatin remodeling proteins with ICP8

In this study, we have used immunoprecipitation and mass spectrometry to identify over 50 cellular and viral proteins that are associated with the herpes simplex virus 1 (HSV-1) ICP8 single-stranded DNA-binding protein. Many of the coprecipitating cellular proteins are known members of large cellular complexes involved in (i) DNA replication or damage repair, including RPA and MSH6; (ii) nonhomologous and homologous recombination, including the catalytic subunit of the DNA-dependent protein kinase, Ku86, and Rad50; and (iii) chromatin remodeling, including BRG1, BRM, hSNF2H, BAF155, mSin3a, and histone deacetylase 2. It appears that DNA mediates the association of certain proteins with ICP8, while more direct protein-protein interactions mediate the association with other proteins. A number of these proteins accumulate in viral replication compartments in the infected cell nucleus, indicating that these proteins may have a role in viral replication. WRN, which functions in cellular recombination pathways via its helicase and exonuclease activities, is not absolutely required for viral replication, as viral yields are only very slightly, if at all, decreased in WRN-deficient human primary fibroblasts compared to control cells. In Ku70-deficient murine embryonic fibroblasts, viral yields are increased by almost 50-fold, suggesting that the cellular nonhomologous end-joining pathway inhibits HSV replication. We hypothesize that some of the proteins coprecipitating with ICP8 are involved in HSV replication and may give new insight into viral replication mechanisms.     

Herpesvirus protein ICP27 switches PML isoform by altering mRNA splicing

Viruses use alternative splicing to produce a broad series of proteins from small genomes by utilizing the cellular splicing machinery. Since viruses use cellular RNA binding proteins for viral RNA processing, it is presumable that the splicing of cellular pre-mRNAs is affected by viral infection. Here, we showed that herpes simplex virus type 2 (HSV-2) modifies the expression of promyelocytic leukemia (PML) isoforms by altering pre-mRNA splicing. Using a newly developed virus-sensitive splicing reporter, we identified the viral protein ICP27 as an alternative splicing regulator of PML isoforms. ICP27 was found to bind preferentially to PML pre-mRNA and directly inhibit the removal of PML intron 7a in vitro. Moreover, we demonstrated that ICP27 functions as a splicing silencer at the 3′ splice site of the PML intron 7a. The switching of PML isoform from PML-II to PML-V as induced by ICP27 affected HSV-2 replication, suggesting that the viral protein modulates the splicing code of cellular pre-mRNA(s) governing virus propagation.     

Deubiquitinating function of adenovirus proteinase

The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.     

Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites

Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication.

Authors Summary

Cellular proteins and cellular membranes are usurped by positive-stranded RNA viruses to assemble viral replicase complexes required for their replication. Tombusviruses, which are small RNA viruses of plants, depend on sterol-rich membranes for replication. The authors show that the tombusviral replication protein binds to cellular oxysterol-binding ORP proteins. Moreover, the endoplasmic reticulum resident cellular VAP proteins also co-localize with viral replication proteins. These protein interactions likely facilitate the formation of membrane contact sites that are visible in cells replicating tombusvirus RNA. The authors also show that sterols are recruited and enriched to the sites of viral replication. In vitro replication assay was used to show that sterols indeed stimulate tombusvirus replication. In summary, tombusviruses use subverted cellular proteins to build sterol-rich membrane microdomain to promote the assembly of the viral replicase complex. The paper connects efficient virus replication with cellular lipid transport and membrane structures.     

The YLDL sequence within Sendai virus M protein is critical for budding of virus-like particles and interacts with Alix/AIP1 independently of C protein

For many enveloped viruses, cellular multivesicular body (MVB) sorting machinery has been reported to be utilized for efficient viral budding. Matrix and Gag proteins have been shown to contain one or two L-domain motifs (PPxY, PT/SAP, YPDL, and FPIV), some of which interact specifically with host cellular proteins involved in MVB sorting, which are recruited to the viral budding site. However, for many enveloped viruses, L-domain motifs have not yet been identified and the involvement of MVB sorting machinery in viral budding is still unknown. Here we show that both Sendai virus (SeV) matrix protein M and accessory protein C contribute to virus budding by physically interacting with Alix/AIP1. A YLDL sequence within the M protein showed L-domain activity, and its specific interaction with the N terminus of Alix/AIP1(1-211) was important for the budding of virus-like particles (VLPs) of M protein. In addition, M-VLP budding was inhibited by the overexpression of some deletion mutant forms of Alix/AIP1 and depletion of endogenous Alix/AIP1 with specific small interfering RNAs. The YLDL sequence was not replaceable by other L-domain motifs, such as PPxY and PT/SAP, and even YPxL. C protein was also able to physically interact with the N terminus of Alix/AIP1(212-357) and enhanced M-VLP budding independently of M-Alix/AIP1 interaction, although it was not released from the transfected cells itself. Our results suggest that the interaction of multiple viral proteins with Alix/AIP1 may enhance the efficiency of the utilization of cellular MVB sorting machinery for efficient SeV budding.     

Interaction Network of Proteins Associated with Human Cytomegalovirus IE2-p86 Protein during Infection: A Proteomic Analysis

Human cytomegalovirus protein IE2-p86 exerts its functions through interaction with other viral and cellular proteins. To further delineate its protein interaction network, we generated a recombinant virus expressing SG-tagged IE2-p86 and used tandem affinity purification coupled with mass spectrometry. A total of 9 viral proteins and 75 cellular proteins were found to associate with IE2-p86 protein during the first 48 hours of infection. The protein profile at 8, 24, and 48 h post infection revealed that UL84 tightly associated with IE2-p86, and more viral and cellular proteins came into association with IE2-p86 with the progression of virus infection. A computational analysis of the protein-protein interaction network indicated that all of the 9 viral proteins and most of the cellular proteins identified in the study are interconnected to varying degrees. Of the cellular proteins that were confirmed to associate with IE2-p86 by immunoprecipitation, C1QBP was further shown to be upregulated by HCMV infection and colocalized with IE2-p86, UL84 and UL44 in the virus replication compartment of the nucleus. The IE2-p86 interactome network demonstrated the temporal development of stable and abundant protein complexes that associate with IE2-p86 and provided a framework to benefit future studies of various protein complexes during HCMV infection.     

Isolation of enzymatically active replication complexes from feline calicivirus-infected cells

A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative "3A-like" protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication.     

Analysis of poliovirus protein 3A interactions with viral and cellular proteins in infected cells

Poliovirus proteins 3A and 3AB are small, membrane-binding proteins that play multiple roles in viral RNA replication complex formation and function. In the infected cell, these proteins associate with other viral and cellular proteins as part of a supramolecular complex whose structure and composition are unknown. We isolated viable viruses with three different epitope tags (FLAG, hemagglutinin [HA], and c-myc) inserted into the N-terminal region of protein 3A. These viruses exhibited growth properties and characteristics very similar to those of the wild-type, untagged virus. Extracts prepared from the infected cells were subjected to immunoaffinity purification of the tagged proteins by adsorption to commercial antibody-linked beads and examined after elution for cellular and other viral proteins that remained bound to 3A sequences during purification. Viral proteins 2C, 2BC, 3D, and 3CD were detected in all three immunopurified 3A samples. Among the cellular proteins previously reported to interact with 3A either directly or indirectly, neither LIS1 nor phosphoinositol-4 kinase (PI4K) were detected in any of the purified tagged 3A samples. However, the guanine nucleotide exchange factor GBF1, which is a key regulator of membrane trafficking in the cellular protein secretory pathway and which has been shown previously to bind enteroviral protein 3A and to be required for viral RNA replication, was readily recovered along with immunoaffinity-purified 3A-FLAG. Surprisingly, we failed to cocapture GBF1 with 3A-HA or 3A-myc proteins. A model for variable binding of these 3A mutant proteins to GBF1 based on amino acid sequence motifs and the resulting practical and functional consequences thereof are discussed.     

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non‐structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1‐4 does not block cellular protein synthesis in BHK cells. Trans‐complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co‐expression of nsP1‐4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T‐cell intracellular antigen and polypyrimidine tract‐binding protein is clearly detected in SINV‐infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut‐off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut‐off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR^?/? mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.     

Proteins associated with purified human cytomegalovirus particles.

Virion-associated proteins isolated from purified human cytomegalovirus particles (strain AD169) were used as substrates for chemical sequence analysis. Extracellular virions, noninfectious enveloped particles, and dense bodies were purified by negative viscosity-positive density gradient centrifugation, and their component proteins were separated by denaturing polyacrylamide gel electrophoresis. The deduced amino acid sequence of individual protein bands was used to identify six corresponding viral genes whose products have not previously been identified as virion constituents: UL47, UL25, UL88, UL85, UL26, and UL48.5. In addition, a 45-kDa cellular protein was identified, and the protein fragments sequenced have a high degree of amino acid identity with actin. However, antiactin monoclonal and polyclonal antibodies did not react with a specific protein in the virus preparations, suggesting that this 45-kDa protein is an immunologically distinct isoform of actin. The newly identified viral and cellular proteins were resistant to protease treatment of purified virions, suggesting that they are unlikely to be contaminants of the viral preparations.